Division of Genetics and Physiology, Department of Biology, University of Turku, Vesilinnantie 5, 20014 Turun Yliopisto, Finland.
J Virol. 2010 Apr;84(7):3666-81. doi: 10.1128/JVI.01340-09. Epub 2010 Jan 20.
Coxsackievirus A9 (CAV9) is a member of the human enterovirus B species within the Enterovirus genus of the family Picornaviridae. It has been shown to utilize alphaV integrins, particularly alphaVbeta6, as its receptors. The endocytic pathway by which CAV9 enters human cells after the initial attachment to the cell surface has so far been unknown. Here, we present a systematic study concerning the internalization mechanism of CAV9 to A549 human lung carcinoma cells. The small interfering RNA (siRNA) silencing of integrin beta6 subunit inhibited virus proliferation, confirming that alphaVbeta6 mediates the CAV9 infection. However, siRNAs against integrin-linked signaling molecules, such as Src, Fyn, RhoA, phosphatidylinositol 3-kinase, and Akt1, did not reduce CAV9 proliferation, suggesting that the internalization of the virus does not involve integrin-linked signaling events. CAV9 endocytosis was independent of clathrin or caveolin-1 but was restrained by dynasore, an inhibitor of dynamin. The RNA interference silencing of beta2-microglobulin efficiently inhibited virus infection and caused CAV9 to accumulate on the cell surface. Furthermore, CAV9 infection was found to depend on Arf6 as both silencing of this molecule by siRNA and the expression of a dominant negative construct resulted in decreased virus infection. In conclusion, the internalization of CAV9 to A549 cells follows an endocytic pathway that is dependent on integrin alphaVbeta6, beta2-microglobulin, dynamin, and Arf6 but independent of clathrin and caveolin-1.
柯萨奇病毒 A9（CAV9）是肠道病毒 B 属人肠道病毒科小核糖核酸病毒属的成员。已证实其利用αV 整联蛋白，尤其是αVβ6，作为其受体。CAV9 与细胞表面初始附着后进入人细胞的内吞途径目前尚不清楚。在这里，我们对 CAV9 进入 A549 人肺癌细胞的内化机制进行了系统研究。整合素β6 亚基的小干扰 RNA（siRNA）沉默抑制了病毒增殖，证实了αVβ6 介导了 CAV9 感染。然而，针对整合素连接信号分子（如Src、Fyn、RhoA、磷酸肌醇 3-激酶和 Akt1）的 siRNA 并不能减少 CAV9 的增殖，这表明病毒的内化不涉及整合素连接的信号事件。CAV9 的内吞作用不依赖于网格蛋白或小窝蛋白-1，但被dynasore 抑制，dynasore 是一种肌球蛋白 V 的抑制剂。β2-微球蛋白的 RNA 干扰沉默有效地抑制了病毒感染，并导致 CAV9 在细胞表面积聚。此外，发现 CAV9 感染依赖于 Arf6，因为通过 siRNA 沉默该分子和表达显性负构象都会导致病毒感染减少。总之，CAV9 进入 A549 细胞的内化途径依赖于整联蛋白αVβ6、β2-微球蛋白、肌球蛋白 V 和 Arf6，但不依赖于网格蛋白和小窝蛋白-1。
